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Doug Chung, JooYeon Jung, Marco Morselli, Matteo Pellegrini, Anthony J Aldave; Posterior polymorphous corneal dystrophy: Transcriptomic impact of OVOL2 and GRHL2 overexpression on corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1450.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the promoter regions of the ovo like zinc finger 2 (OVOL2) and grainyhead like transcription factor 2 (GRHL2) genes are associated with posterior polymorphous corneal dystrophy (PPCD), an inherited disorder affecting the corneal endothelium, and have been demonstrated to be associated with ectopic expression of their respective gene. To determine the impact of OVOL2 and GRHL2 overexpression on the transcriptome of corneal endothelial cells, RNA-sequencing analysis was performed on primary human corneal endothelial cells transduced with lentivirus carrying either OVOL2 or GRHL2 overexpression vectors.
Lentiviruses containing OVOL2, GRHL2, or empty (as a control) overexpression constructs were generated, characterized, and then transduced into primary human corneal endothelial cells (pHCEnC). Total RNA and protein lysates were isolated from transduced pHCEnC. Western blot was performed to confirm overexpression of OVOL2 and GRHL2. RNA sequencing and transcriptomic analyses were performed, including differential gene expression, gene ontology (GO) enrichment, and canonical pathway analyses.
OVOL2 overexpression in pHCEnC led to a total of 576 differentially expressed genes (DEG) (|fold-change| ≥ 2, q-value ≤ 0.05, min group expression ≥1 RPKM), with 388 upregulated and 188 downregulated genes, compared to controls. Additionally, OVOL2 overexpression in pHCEnC led to a -2.17 fold-change in ZEB1. GRHL2 overexpression in pHCEnC caused differential expression of 443 genes, with 225 upregulated and 218 downregulated genes, compared to controls. Overexpression of GRHL2 caused a fold-change of -1.46 in ZEB1. In both OVOL2 and GRHL2 overexpression, gene ontology and canonical pathway analyses identified cell adhesion, cell signaling, and immune response pathways to be in significantly impacted.
Overexpression of either OVOL2 or GRHL2 caused the downregulation of ZEB1 in human corneal endothelial cells, which supports the hypothesis that PPCD-associated OVOL2 and GRHL2 promoter mutations lead to PPCD by inducing ectopic expression of OVOL2 and GRHL2, respectively, and subsequently repressing the expression of ZEB1, a gene well-established to be associated with PPCD.
This is a 2020 ARVO Annual Meeting abstract.
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